This project will continue investigation of the molecular events that determine the mechanism of steroid hormone binding to specific macromolecular binding sites. New affinity laveling steroids will be synthesized for these studies. Their effect on pregnancy will be tested in rats and sheep toilluminate the connection between the mechanism of steroid binding to macromolecules and the mechanism of steroid hormone action. Ovine fetal erythrocy te 20 Alpha-hydroxysteroid oxidoreductase (20 Alpha-HSD) will be prepared in quantity according to our newly established method established method for isolating 20 Alpha-hydroxysteroid oxidoreductase (EC 1.1.1.149) from bovine fetal erythrocytes. Both ovine and bovine 20 Alpha-HSD will be reacted with new adenosine and progesterone derivatives containing 3H and 14C bromoacetate side chains to characterize the active site amino acid compositions. The nucleosides will be used to continue our labeling of the cofactor binding region, and the steroids will be used for the steroid binding region of the active sties. Antibodies against ovine 20 Alpha-HSD will be raised in rabbits and used to measure levels of ovine fetal 20 Alpha-HSD in erythrocytes throughout pregnancy in cannulated sheep. By obtaining a profile for fetal 20 Alpha-HSD levels during gestation and also inactivating the enzyme in vivo with appropriate affinity labeling steroids we hope to learn the role of 20 Alpha-HSD in pregnancy. Th same antibodies will be used to measure conformational changes in 20 Alpha-HSD accompanying binding or progesterone or progesterone-protein conjugates to the enzyme. We will continue experiments with 17Beta-estradiol-7Alpha-butanoic acid - (rat) albumin in vivo to learn the precise mechanism of its uterotropic activity. Particular attention will be focused on evaluating the ability of the estrogen-protein conjugate to cross the target cell membrane and enter the nucleus. Definition of the mechanisms of steroid hormone binding to macromolecular binding sites of enzymes and receptor protein systems is the central goal of the project. This will braoden and deepen our understanding of the relationship between steriod structure and hormone action, leading to the design of new steroids for control of the mammalian reproductive system.